U.S. patent number 5,077,215 [Application Number 07/360,887] was granted by the patent office on 1991-12-31 for neutralized perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride copolymer surface for attachment and growth of animal cells.
This patent grant is currently assigned to Commonwealth Scientific and Industrial Research Organisation, Telectronics Pty. Limited. Invention is credited to Graham Johnson, Brian R. McAuslan, William Norris, John G. Steele.
United States Patent |
5,077,215 |
McAuslan , et al. |
December 31, 1991 |
Neutralized perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl
fluoride copolymer surface for attachment and growth of animal
cells
Abstract
Neutralized surface of a polymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride for
attachment and growth of animal cells in vivo or in vitro, the
comonomer preferably being tetrfluorethylene.
Inventors: |
McAuslan; Brian R. (Clareville,
AU), Steele; John G. (North Rocks, AU),
Norris; William (North Sydney, AU), Johnson;
Graham (Peakhurst, AU) |
Assignee: |
Telectronics Pty. Limited (New
South Wales, AU)
Commonwealth Scientific and Industrial Research Organisation
(Campbell, AU)
|
Family
ID: |
3772454 |
Appl.
No.: |
07/360,887 |
Filed: |
July 17, 1989 |
PCT
Filed: |
September 19, 1988 |
PCT No.: |
PCT/AU88/00368 |
371
Date: |
July 17, 1989 |
102(e)
Date: |
July 17, 1989 |
PCT
Pub. No.: |
WO89/02457 |
PCT
Pub. Date: |
March 23, 1989 |
Foreign Application Priority Data
Current U.S.
Class: |
424/423; 435/402;
623/920; 623/915 |
Current CPC
Class: |
C12N
5/0068 (20130101); Y10S 623/92 (20130101); Y10S
623/915 (20130101); C12N 2533/30 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); C12M 3/04 (20060101); C12N
005/00 (); A61F 002/02 (); A61F 002/04 (); A61F
002/06 () |
Field of
Search: |
;435/240.23,240,243
;600/36 ;623/1 ;424/423 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
|
8665675 |
|
Nov 1975 |
|
AU |
|
8671482 |
|
Jan 1983 |
|
AU |
|
9184082 |
|
Apr 1983 |
|
AU |
|
5014085 |
|
May 1986 |
|
AU |
|
6048386 |
|
Feb 1987 |
|
AU |
|
0092302 |
|
Dec 1983 |
|
EP |
|
2724200 |
|
Dec 1978 |
|
DE |
|
55-160029 |
|
Dec 1980 |
|
JP |
|
62-288614 |
|
Dec 1987 |
|
JP |
|
1311370 |
|
Mar 1973 |
|
GB |
|
Other References
Penner, et al., Ion Transporting Composite Membranes 1.
Nafion-Impregnated Gore-Tex Journal of the Electrochemical Society,
vol. 132, pp. 514-515, 1985. .
Hynes Molecular Biology of Fibronectin. Annual Review of Cell
Biology, vol. 1, pp. 67-90, 1985. .
Adhesion of Cells to Polystyrene Surfaces, by A. S. G. Curtis et
al., Departments of Cell Biology and Chemistry, University of
Glasgow, Glagow G12 8QQ Scotlan, UK. the Journal of Cell Biology,
vol. 97, Nov. 1983, pp. 1500-1506. .
Attachment and growth of BHK cells and liver cells on polystyrene:
Effect of surface groups introduced by treatment with chromic acid,
H. G. Klemperer and P. Knox, Dept. of Biochemistry and Dept. of
Cancer Studies, University of Birmingham Lab. Practice, vol. 26,
No. 3. .
Substrate Hydroxylation and Cell Adhesion, A. S. G. Curtis et al.,
J. Cell Sci. 86, 9-24 (1986) The Company of Biologist Ltd.
Department of Cell Biology, University of Glasgow, Glasgow, UK.
.
Cellular interactions with synthetic polymer surfaces in culture M.
J. Lydon et al.,. Unilever Research Laboratory, Colworth House,
Shambrook, Bedfordshire, MK44 1LQ, UK, 1985 Butterwoth & Co.
(publilshers) Ltd., Biomaterials 1985, vol. 6 Nov. .
Coating Bacteriological Dishes With Fibronectin Permits Spreading
and Growth of Human Diploid Fibroblasts by Frederick Grinnell and
Jannet L. Marshall, Department of Cell Biology, University of Texas
Health Science Center, Cell Bio. Intern. Reports. vol. 6, No. 11,
11/82. .
Adhesion and Spreading of Cells on Charged Surfaces, by N. G.
Maroudas, J. Theor., Biol. (1975) 49, pp. 417-424-Imperial Cancer
Research Fund Laboratories, London England. .
Sulphonated Polystyrene as an Optimal Substratum for the Adhesion
and Spreading of Mesenchymal Cell in Monovalent and Divalent Saline
Solutions by N. G. Maroudas; -Cell Phsiol 90: 511-520, Mar.,
1977..
|
Primary Examiner: Stone; Jacqueline
Assistant Examiner: Elliott; George C.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt
Claims
We claim:
1. In a prosthesis or sponge implantable in a body, the improvement
comprising forming the surface of said prosthesis or sponge from a
neutralized copolymer of perfluoro-3,6-dioxa-4-methyl-7-octene
sulphonyl fluoride and a monomer.
2. The prosthesis or sponge of claim 1 wherein the monomer is
tetrafluoroethylene.
3. The prosthesis or sponge of claim 1 further comprising a
supporting material to which said copolymer is applied.
4. The prosthesis or sponge of claim 3 wherein said supporting
material is selected from the group consisting of a polymer,
ceramic, metal, glass and preformed membrane.
5. The prosthesis or sponge of claim 4 wherein said polymer is a
porous polymer.
6. The prosthesis or sponge of claim 5 wherein said porous polymer
is polytetrafluoroethylene or expanded polytetrafluoroethylene.
7. The prosthesis or sponge of claim 5 wherein the porous polymer
is knitted or woven polyester.
8. The prosthesis or sponge of claim 5 wherein the porous polymer
is polyurethane.
9. The prosthesis of sponge of claim 1 in the form of a tube.
10. The prosthesis or sponge of claim 1 wherein the surface further
comprises adsorbed adhesive proteins.
11. The prosthesis or sponge of claim 10 wherein said adhesive
proteins are derived from serum.
12. The prosthesis or sponge of claim 11 wherein said adhesive
serum proteins are selected from the group consisting of
fibronectin, vitronectin, thrombospondin and adhesive fragments of
any of these proteins.
13. The prosthesis or sponge of claim 1 wherein the surface further
comprises animal cells adhered thereto.
14. A surface for the attachment and growth of animal cells in
vivo, said surface comprising the neutralized form of a copolymer
of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and
monomer with animal cells adhered thereto.
15. A process for the preparation of a surface for the attachment
and growth of animal cells in vivo, said process comprising
applying a copolymer of perfluoro-3-, 6-dioxa-4-methyl-7-octene
sulphonyl fluoride and a monomer to an appropriate substrate,
neutralizing the resultant surface and adhering animal cells to the
surface.
16. A process for the attachment and growth of animal cells in vivo
comprising: exposing animal cells to a prosthesis or sponge having
a surface formed from a neutralized copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer.
17. A process for the attachment and growth of animal cells in
vitro comprising: exposing animal cells in vitro to a surface
formed from a neutralized copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer.
18. The process of claim 17 which further comprises exposing said
surface to a medium containing animal cells and adhesive proteins.
Description
FIELD OF THE INVENTION
This invention relates to the use of a copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer as a surface for the attachment and growth of adherent
animal cells. The invention has particular application to the
manufacture and use of prosthetic vascular grafts, connective
tissue replacements and soft tissue replacements that incorporate
such a copolymer.
BACKGROUND ART
The design or selection of materials useful in vascular prostheses
requires an understanding of the characteristics necessary for
irreversible endothelialisation of a surface and for inhibition of
undesirable platelet interactions. An approach to the development
of vascular prostheses that has been taken has been guided by the
object of circumventing the acute problems of platelet activation,
adhesion and thrombogenesis. This approach involves designing a
blood interface which disallows thrombogenesis by preventing
platelet activation directly, and may be achieved either by the
selective incorporation or adsorption of platelet binding
inhibitors, such as serum albumin or heparin, or by providing a
surface which directly repels or inactivates platelets
electrostatically. However these modifications might also suppress
the attachment and growth of endothelial cells on the luminal
surface of the prosthesis. Grafts prepared using this approach may
therefore be regarded as unhealed and a physiological and
anatomical state comparable to the normal luminal structure is not
achieved.
It is generally known that surfaces which support endothelial cell
growth comparable to that seen on glow discharged polystyrene also
tend to be thrombogenic. However it is also known that sulphonated
polystyrenes have antithrombogenic activity which is reported to be
a feature of the negative charge of sulphonate groups. The present
invention has been developed by following this line of
investigation.
In a recent study, McAuslan and Johnson [(1987) J. Biomedical
Materials Research 21.921-935] showed that the hydroxyl rich
surface of poly(hydroxyl ethyl methacrylate)(pHEMA) hydrogel can be
converted from a non-cell adhesive to a highly cell adhesive state
by either hydrolytic surface etching or by copolymerization with
methacrylic acid. Thus cell adhesion appeared to correlate with the
introduction of surface COOH groups although this alone was not a
sufficient condition. This has raised the question of whether other
negatively charged moieties would be just as effective at promoting
cell attachment.
A fluorocarbon polymer with pendant sulphonic groups is the
chemically inert, non-crosslinked cation-exchange resin known by
the trade mark NAFION. NAFION is chemically identified as a
copolymer of tetrafluoroethylene and
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride. The
mechanical and chemical stability of this perfluorosulphonate
ionomer and its selective permeability to charged ions had made it
useful for industrial electrochemical separating processes. It can
be prepared as films or tubes and is hydrophilic, which is in
contrast to polytetrafluoroethylene (PTFE, which is known by the
trade mark TEFLON) or expanded PTFE (which is known by the trade
mark GORE-TEX), a material which is in wide use as a vascular
graft.
We have now found that any copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer, and particularly NAFION, may, when in a neutralised form,
be used as a surface for the attachment and growth of adherent
animal cells from different tissue sources, including endothelial
cells. In this specification and claims, reference to being in a
neutralised form means within one pH unit of pH 7.0.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a material
useful in vascular prostheses and other implantables having
improved biocompatibility arising from enhanced endothelial cell
attachment properties and anti-thrombogenicity which will
substantially overcome the disadvantages of the prior art.
In accordance with one aspect of the present invention, there is
provided a surface for the attachment and growth of cells in vivo,
said surface comprising the neutralized form of a copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer. Preferably, the monomer is tetrafluoroethylene. The above
surface may be adsorbed or attached to an appropriate substrate
that is preferably porous. The types of substrate that may be used
include polymers, ceramics, metals, glass or preformed membranes.
When a polymer substrate is used, the polymer is preferably porous
such as polytetrafluoroethylene, expanded polytetrafluoroethylene,
knitted or woven polyester and polyurethane.
In a preferred form, the surfaces of the present invention that may
be used in vivo are in the form of a sponge or tube and may be
adapted for use in a biosensor. The surfaces of the present
invention that may be used in vivo may also be modified by
selective incorporation of platelet binding inhibitors such as
serum albumen or heparin or by treating the surfaces with agents
that specifically repel or inactivate platelet attachment.
In accordance with another aspect of the present invention, there
is provided a surface for the attachment and growth of cells in
vivo, said surface comprising the neutralised form of a copolymer
of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer optionally adsorbed or attached to an appropriate substrate
as described above, and wherein said surface further includes
adsorbed adhesive proteins. The preferred adhesive proteins are
derived from serum and include fibronectin, vitronectin or adhesive
fragments of these proteins. Other preferred adhesive proteins
include laminin, collagens and thrombospondin and adhesive
fragments of these proteins. The above surfaces that may be used in
vivo, may also have adsorbed thereto adhesive proteins or their
adhesive fragments.
In a further preferred embodiment, the above surfaces for use both
in vivo and in vitro include adhered cells of the type sought to be
grown.
In accordance with yet another aspect of the present invention,
there is provided a process for the preparation of a surface for
the attachment and growth of cells in vivo, said process comprising
applying a copolymer of perfluoro-3,6-dioxa-4-methyl-7-octene
sulphonyl fluoride and a monomer to an appropriate substrate. The
surface so prepared must be brought to neutrality, and this may be
done by either neutralizing the resultant surface or by applying
the above copolymer in a neutralized form to the appropriate
substrate.
The above copolymer is preferably applied to the substrate by means
of radiation grafting or adhesive bonding.
In accordance with a further aspect of the present invention, there
is provided a process for the preparation of a surface for the
attachment and growth of cells in vitro, said process comprising
exposing adhesive proteins or adhesive serum proteins to a surface
comprising the neutralized form of a copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer optionally adsorbed or attached to an appropriate
substrate, whereupon the copolymer adsorbs said proteins to form a
copolymer-protein complex.
In a further preferred embodiment, the surfaces prepared according
to the above processes for use both in vivo and in vitro are
further exposed to cells of the type sought to be grown, whereupon
the cells adhere to the said surface to further improve its cell
attachment and growth properties.
In accordance with a still further aspect of the present invention,
there is provided a method for the attachment and growth of cells
to a surface both in vivo and in vitro, which method comprises
exposing cells or a medium containing cells and adhesive proteins
to a surface of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the relative growth rates of bovine aortal
endothelial cells on the following surfaces: tissue culture plastic
(TCP), NAFION 22, 70 and 125 films prepared as described in Example
1, during the period of 4 days after seeding, and,
FIG. 2 (A and B) shows the difference in cell morphology and cell
density of bovine aortal endothelial cells after 4 days culture on
untreated TEFLON (FIG. 2A) or on TEFLON that was coated with
NAFION, as described in Example 1, (FIG. 2B). Cells were stained
with acridine orange and photographed under UV light and the
photographs are at 80 times magnification.
FIG. 3a is a graph of the relative growth rate of the human cell
line Hep-2 on the following surfaces: tissue culture plastic,
NAFION 70 prepared as described in Examples 1 and 3 and both the
above surfaces precoated with fibronectin prior to cell seeding,
cultured for a period of 7 days.
FIG. 3b is a graph of the relative growth rate of the human cell
line HeLa on the following surfaces: tissue culture plastic, NAFION
70 prepared as described in Examples 1 and 3 and both the above
surfaces precoated with fibronectin prior to cell seeding, cultured
for a period of 8 days.
FIG. 3c is a graph of the relative growth rate of the human cell
line HT 1080 on the following surfaces: tissue culture plastic,
NAFION 70 prepared as described in Examples 1 and 3 and both the
above surfaces precoated with fibronectin prior to cell seeding,
cultured for a period of 7 days.
FIG. 4 (A-D) shows the difference in morphology of human umbilical
arterial endothelial cells cultured on tissue culture plastic (FIG.
4A and FIG. 4C) or NAFION 70, prepared as described in Examples 1
and 3 (FIG. 4B and FIG. 4D). Some of the surfaces (FIG. 4C and FIG.
4D) were precoated with fibronectin prior to cell seeding. The
cells were cultured for 7 days then photographed. The photographs
in FIG. 4A and FIG. 4B are at 110 times magnification and in FIG.
4C and FIG. 4D, at 220 times magnification. Note that the
morphology of the cells on NAFION 70 is indistinguishable from that
of the cells on the tissue culture plastic, and not also the
enhanced cell spreading on each of the surfaces when precoated with
fibronectin.
FIG. 5a is a graph of the relative growth rates of the human
umbilical arterial endothelial cells cultured for 7 days on tissue
culture plastic (TCP) and TCP precoated with fibronectin prior to
cell seeding.
FIG. 5b is a graph of the relative growth rates of the human
umbilical arterial endothelial cells cultured for 7 days on TEFLON
that was coated with NAFION (NAF) as described in Example 1, and
NAF precoated with fibronectin prior to cell seeding.
FIG. 5c is a graph of the relative growth rates of the human
umbilical arterial endothelial cells cultured for 7 days on
untreated TEFLON (TEF), and TEF precoated with fibronectin prior to
cell seeding.
FIG. 6 (A-F) shows the difference in morphology of human umbilical
arterial endothelial cells cultured on GORE-TEX (FIG. 6A and FIG.
6B), or GORE-TEX that was coated with NAFION as described in
Example 3 (FIG. 6C and FIG. 6D). Another sample of NAFION-coated
GORE-TEX (FIG. 6E and FIG. 6F) was precoated with fibronectin prior
to cell seeding. The cell attachment and morphology was examined by
scanning electron microscopy and the magnification of each panel is
given as follows: for FIG. 6A, FIG. 6C and FIG. 6E, 200 microns=62
mm on the print, whereas for FIG. 6B, FIG. 6D and FIG. 6F, 50
microns=61 mm on the print. Note the sparce cell attachment to
GORE-TEX (FIG. 6A and FIG. 6B) but markedly better cell coverage
and spreading on the GORE-TEX that was coated with NAFION, giving
almost complete cell coverage of the surface (FIG. 6C to FIG. 6F).
The cells were fixed after 1 day of culture (FIGS. 6A, 6B, 6C, 6D,
6F) or 3 days of culture (FIG. 6E).
FIG. 7 is a diagram of the apparatus used to test cellular
attachment under conditions of flowing culture medium, as described
in Example 4.
DESCRIPTION OF PREFERRED EMBODIMENTS
In order that the invention may be more readily understood and put
into practical effect, reference will now be made to the following
examples that describe the use of NAFION as a preferred embodiment
of the copolymer of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl
fluoride and a monomer.
EXAMPLE 1
Preparation of NAFION N117, NAFION N125, NAFION 22 and NAFION 70
membrane preparations, and PTFE and GORE-TEX coated with NAFION
70.
Portions of NAFION N117 membrane and NAFION N125 membrane were cut
into 1 cm.sup.2 pieces and washed in acetone followed by absolute
ethanol. The pieces were then treated with 0.2% EDTA to remove
cationic contaminants, washed thoroughly with deionised water and
sterilised by autoclaving. Prior to tissue culture studies the
pieces were extensively washed in sterile phosphate buffered saline
(PBS) pH 7.2.
A 5% (w/vol) solution of NAFION 1100 Equivalent Weight
perfluorinated ion-exchange resin was used for casting the
following membranes:
i) NAFION 22--prepared by casting 0.5 ml 5% NAFION solution in the
lid of a 35 mm diameter tissue culture petri dish as 22.degree.
C.
ii) NAFION 70--prepared by repeating the procedure for NAFION 22 at
70.degree. C. for 2 hours.
Prior to tissue culture studies, the NAFION 22 and NAFION 70
membranes were sterilised under ultraviolet light for 2 hours and
then extensively washed in serum free tissue culture medium.
iii) Unfilled virgin TEFLON samples were washed extensively in
ethanol and some were coated with NAFION solution and some were
left uncoated, for cell culture studies. Approximately 1 cm.sup.2
pieces of material were coated with 50 microliters of 5% NAFION
Equivalent Weight 1100 solution and treated at 70.degree. C. for 2
hours. Substrates so prepared and tissue culture polystyrene (TCP)
were sterilised under UV light for 2 hours and washed extensively
in serum free tissue culture medium before being used for cell
culture studies.
Equilibrium water content (EWC)
The EWC of both NAFION N125 and the NAFION 22 and 70 membranes was
determined essentially as described by Pedley and Tighe ](1979) Br.
Polym. J., 11, 130-135]. In both cases the NAFION was pretreated
with 0.2% EDTA and then washed and equilibrated in deionised water
for 4 to 6 days before weighing.
Results
Commercially available preformed sheet NAFION (NAFION N-117 and
N-125) and NAFION membrane cast from a 5% solution on either glow
discharged or non-glow discharged polystyrene at 22.degree. C.
(NAFION 22) were transparent neutral coloured substrates. After
casting at 70.degree. C. (NAFION 70), no difference in texture of
colour was observed. However this treatment rendered it insoluble
in ethanol and acetone. NAFION membranes were cast in a variety of
thickness from approximately 10 to 40 microns. By comparison the
preformed sheet NAFION used in this study was approximately 100
microns thick. NAFION N125 has an equilibrium water content of
12.0% and NAFION 22 and 70 equilibrium water contents of 32% and
36% respectively. NAFION membranes cast on polystyrene could be
peeled off the surface by gently pulling with forceps and the
thickness of the membrane determined the fragility of such
material.
EXAMPLE 2
Attachment and Growth of Bovine Endothelial Cells and other
Adherent Animal Cells on NAFION
Methods
Cell Culture and Cell Growth Rate Determination
A clonal line of normal bovine aortic endothelial (BAE) cells were
grown and maintained in M199 cell culture medium supplemented with
20% (v/v) fetal calf serum. To determine the increase in the number
of cells growth on the different substrates, 2 ml of M199 cell
culture medium supplemented with 20% foetal bovine serum containing
between 5.times.10.sup.4 and 2.times.10.sup.5 BAE cells were added
to dishes containing NAFION N125, or coated with either NAFION 22
or NAFION 70, and incubated in a humidified atmosphere of 5%
CO.sub.2 in air at 37.degree. C. After 6 hours, cell attachment was
estimated by counting cells within 15 randomly chosen 0.931
mm.sup.2 areas. Each polymer sample was the subject of three
individual trials and mean cell numbers expressed per cm.sup.2.
After this initial period cell numbers were determined every 24
hours to determine cell growth rate. For comparison the growth of
BAE cells on GORE-TEX, TEFLON and glow discharged tissue culture
polystyrene was also determined. Cell morphology was investigated
by routine light microscopy of cultured surfaces for the NAFION
membranes. For the NAFION cast onto TEFLON surfaces, the opacity of
the TEFLON precluded the use of phase contrast microscopy for a
full visual comparison, so cells were fixed with 2.5%
gluteraldehyde, stained with 3 mM acridine orange then photographed
under UV light.
Results
It is known that NAFION is a strong acid and washing the cast
polymers (NAFION 22 and NAFION 70) in serum-free tissue culture
medium showed that a comparatively large volume of medium was
required to neutralise its acidic property. This is therefore, an
important aspect of the use of NAFION as a substrate for cell
growth which must be taken into account in its preparation.
Similarly, NAFION preformed membrane (NAFION N117 and NAFION N125)
required neutralisation before use for cell culture.
A wide range of different cell types have been successfully grown
on NAFION. These include bovine aortic endothelial cells (BAE),
bovine aortic smooth muscle cells, bovine corneal endothelial
cells, bovine retinal capillary endothelial cells, baby hamster
kidney fibroblasts, and 3T3 fibroblasts. All cell types displayed
their own characteristic morphology and growth characteristics when
observed on TCP control dishes. Endothelial cells formed a
cobblestone pavement monolayer whilst fibroblasts showed spindle
shaped morphology and eventually formed a whorl-like pattern.
Since it was proposed that the material might show potential as a
component of a vascular prosthesis the attachment and growth of BAE
cells on NAFION compared to TCP and TEFLON was studied in some
detail. Cell attachment in vitro was found to be dependent on the
presence of serum; and in the absence of serum no cells were
attached to any of the NAFION preparations after six hours. The
attachment of BAE cells to the different substrates after 6 hours
was expressed with respect to cell attachment to tissue culture
polystyrene (which was set as 100%). The BAE cell attachment to
NAFION 70 and NAFION 22 was 109.+-.3.2% (Mean Standard Deviation)
and 97.+-.3.6% respectively; to NAFION N125, 84.+-.3.2% and to
TEFLON, 75.+-.6.8%. Of particular interest is the fact that cells
appeared to be more evenly distributed on the surface of the NAFION
films that on NAFION N-125 or tissue culture polystyrene. FIG. 1
shows the kinetics of growth of BAE cells on NAFION N-125, 22 and
70 compared to their growth on tissue culture polystyrene after 4
days. Only small differences were seen in the growth rates and
final numbers of cells on the NAFION preparations compared with
TCP. The growth rate of BAE cells on NAFION preparations observed
in these experiments was considerably higher than the level
recognised for such cells on the commonly used TEFLON vascular
graft material. Cell growth on NAFION coated TEFLON showed a
similar improvement, over such cells grown on TEFLON alone. By
casual appraisal cells cultured on TEFLON had a slower growth rate
than cells cultured on the other polymers. In contrast to BAE cells
seeded on NAFION, cells seeded on TEFLON failed to achieve the
characteristic polyhedral morphology and remained fibroblastoid
until almost confluent. This effect was demonstrated by growing
10.sup.5 BAE cells/ml in culture for 4 days on untreated TEFLON and
NAFION coated TEFLON. BAE cells grown on untreated TEFLON displayed
a typical patchiness or fibroblast-like morphology in the areas of
sparse cover (FIG. 2a). This is characteristic of BAE cells when
growing on a less than ideal substrate. In contrast, BAE cells
grown on NAFION coated TEFLON achieved the polyhedral morphology
characteristic of such cells grown on ideal substrates (FIG. 2b).
Further, BAE cells could be maintained in culture on NAFION N125
successfully for up to 3 weeks.
The growth of BHK fibroblasts on non-glow discharged polystyrene
was facilitated by coating the surface with NAFION solution. In
accordance with the findings for BAE cells, no difference between
the behaviour of BHK fibroblasts to NAFION 22 compared to glow
discharged polystyrene was seen whereas cells failed to attach and
spread properly or nonglow discharged polystyrene (results not
shown). Attachment of BAE cells to TEFLON was less strong than to
polystyrene or NAFION as seen when only gently pipetting of medium
or PBS with a Pasteur pipette was sufficient to detach the cells
from the TEFLON surface. Such physically weak attachment to tissue
culture polystyrene and NAFION was not seen.
EXAMPLE 3
Attachment and Growth of Human Endothelial Cells and other Human
Cells on NAFION 70 Membrane Preparations.
Methods
In some experiments of this Example, the serum adhesive
glycoprotein fibronectin (Fn) was removed from serum prior to use
of the serum for cell culture by passage over a gelatin-Sepharose
affinity column. Serum treated on a gelatin-Sepharose column was
confirmed to be free of Fn by immunoassay of the Fn content. In
other experiments, the serum adhesive glycoprotein vitronectin (Vn)
was removed by passage over an affinity column consisting of
immobilized anti-Vn antibody. The sera that were depleted in Vn by
this affinity technique were confirmed to have been exhaustively
stripped of vitronectin by immunoassay for Vn content.
Human cell lines HeLa from cervical carcinoma, HeP-2 from carcinoma
of Larynx, and HT 1080 from human fibrosarcoma were grown in a
growth medium consisting of minimal essential medium supplemented
with 10% (v/v) foetal calf serum, 60 microgram/ml penicillin and
100 microgram/ml streptomycin.
A human umbilical artery endothelial (HUAE) cell culture was
established and grown in 75 cm.sup.2 tissue culture polystyrene
(TCP) flasks coated with Fn. Coating with Fn was achieved by
incubating the flasks with 5 ml solution of 40 ug/ml Fn in PBS at
37.degree. C. for 1 hour prior to cell seeding. Excess solution was
removed before cells were added. The cells were routinely
maintained in a growth medium consisting of an equal mixture of
McCoy 5A (modified) and BM86-Wissler media supplemented with 30%
v/v foetal bovine serum, 40 ng/ml fibroblast growth factor, 60
ug/ml endothelial cell growth supplement, 20 ug/ml insulin, 60
ug/ml penicillin and 100 ug/ml streptomycin. The cells were
routinely passaged using trypsin-versene, and for experimental work
cells were used between passage 15 and passage 20 (inclusive).
For attachment and cell growth studies, NAFION 70 films cast onto
22 ml wells were equilibrated with PBS, and 2 ml of growth medium
containing 5.times.10.sup.4 cells was added to each well. The cell
attachment was determined by counting, in randomly selected field,
the total number of cells and the number of these cells that had
spread onto the surface. Cell growth in each well was quantitated
by counting 5 randomly selected fields per well after successive
days of culture, until cell confluence was reached. The mean and
standard error of cell number per cm.sup.2 for triplicate samples
were determined.
GORE-TEX samples were cut into pieces of approximately 1 cm.sup.2,
coated with approximately 0.3 ml NAFION solution per piece, then
immediately treated at 70.degree. C. for 2 hrs. The NAFION-coated
GORE-TEX was exposed to UV light for 2 hrs and then washed
extensively in serum-free tissue culture medium. Some samples were
then coated with 40 ug/ml Fibronectin for 45-60 min at 37.degree.
C. prior to seeding with HUAE cells.
Results
The attachment and growth of human cell lines Hep-2, HeLa and HT
1080 was compared to that on tissue culture polystyrene (TCP). Cell
attachment and growth was also determined on NAFION films that had
been coated with a solution of 40 ug/ml Fibronectin. The human cell
lines Hep-2, HeLa and HT 1080 all attached and grew on NAFION films
(see FIG. 3 for cell growth curves). It was necessary to use
culture medium that contained serum for the cells to attach and to
grow on the NAFION surface. In the case of cell lines Hep-2 and
HeLa, the rate of cell growth was increased where the NAFION film
had been precoated with Fn (see FIG. 3 for comparison of Fn coated
NAFION and NAFION that had not been coated with Fn) whereas in the
case of HT 1080 cells the Fn coating of the NAFION had no positive
effect on the cell growth rate.
HUAE cells were grown on NAFION and compared to growth on TCP. It
was also necessary to use culture medium that contained serum for
the HUAE cells to become attached and to grow. Fn-coated TCP was
also included as a control surface, as this surface is known to
support good HUAE cell attachment and growth. The number of HUAE
cells attached to the NAFION surface as viewed after 4 hours fo
cell seeding was equivalent to that on TCP, whereas for the
Fn-coated NAFION, the number of cells attached was equivalent to
that on the Fn-coated TCP. The morphology of the HUAE cells
attached to the NAFION surface was generally similar to that of the
HUAE cells seeded onto TCP, see FIG. 4. This morphology indicated
that although the HUAE cells had attached to the NAFION surface
when seeded in the presence of serum, the cells had not formed the
well spread morphology that is typical of HUAE cells that have been
seeded onto Fn-coated TCP. However the morphology of the HUAE cells
that attached to the Fn-coated NAFION films was well spread and the
cell morphology was similar to HUAE cells growing of Fn-coated TCP,
see FIG. 4.
HUAE cells grew on the NAFION and Fn-coated NAFION surfaces at a
rate that was similar to the cell growth on TCP and Fn-coated TCP,
respectively (see FIG. 5 for growth curve).
The role that Fn from the serum and Vitronectin from the serum may
play in the attachment of the HUAE cells to the NAFION and
Fn-coated NAFION surfaces was determined by selective removal of
these components from the serum used in the culture medium in which
the cells were seeded. Selective removal of Vn from the culture
medium completely abolished the attachment of HUAE cells to the
NAFION surface. The importance of Vitronectin (which is also known
as serum spreading factor, epibolin or 70K spreading factor) has
been previously reported for other polymer surfaces such as tissue
culture polystyrene, see Grinnell [(1976) Exp. Cell Res., 97,
265-274 and (1977) Exp. Cell Res., 110, 175-190]. Attachment of
HUAE cells to Fn-coated NAFION over a 4 hour period when seeded in
culture medium containing Vn-depleted serum was equivalent to that
of HUAE cells seeded in intact medium onto the Fn-coated
surface.
The selective removal of Fn from the seeding culture medium did not
abolish the attachment of HUAE cells to the NAFION surface. As a
consequence of removal of serum Fn, the rate of cell attachment was
somewhat reduced over the first 4 hours as compared to cell
attachment to NAFION where the culture medium contained intact
serum. However after 24 hours the HUAE cell coverage of the NAFION
surface with the Fn-depleted culture medium was identical to that
seen on the NAFION surface with the medium containing intact serum.
The use of culture medium containing vitronectin-depleted serum for
seeding of HUAE cells onto Fn-coated NAFION surface did not effect
the rate and extent of HUAE cell attachment and cell morphology, as
compared to that where the HUAE cells were seeded onto Fn-coated
NAFION using culture medium containing intact serum.
These results indicate that the serum-dependence of the attachment,
cell spreading and growth of HUAE cells on a NAFION surface that
has not been precoated with purified Fn or other adhesive proteins
involves as an essential component the serum adhesive glycoprotein
Vitronectin. Vitronectin from serum or culture medium containing
serum is known from previous work to adsorb readily onto other
culture surfaces such as tissue culture polystyrene. The results
also indicate that the NAFION surface is similar to other surfaces
used for the attachment of cells in that the adhesive glycoprotein
Fn may be purified from serum and then coated onto the NAFION
surface to give a substratum that supports good HUAE cell
attachment, with consequential effects on cell growth. Taken
together, these results indicate that the adsorption of adhesive
serum proteins such as vitronectin or fibronectin onto a NAFION
surface produces a substratum for promoting cell adhesion and
growth.
GORE-TEX-NAFION-Fn as a substratum for HUAE cell growth
NAFION was coated onto GORE-TEX, then the NAFION GORE-TEX surface
was seeded with HUAE cells. In some samples the NAFION-GORE-TEX
surface was precoated with Fibronectin prior to cell seeding. The
HUAE cells attached to the NAFION-GORE-TEX surface and grew to
produce a surface that was almost completely covered with HUAE
cells (See FIG. 6). The HUAE cells grown on each of the
NAFION-GORE-TEX surface and the fibronectin-coated nation-GORE-TEX
surface had a well attached and spread morphology as observed in
the scanning electron microscope (see FIG. 6).
EXAMPLE 4
Attachment and Growth of Ovine Endothelial Cells on NAFION
tubes.
Methods
An ovine carotid arterial endothelial (OCAE) cell culture was
established after the methodology of Jaffe ((ed) Biology of
Endothelial Cells (Developments in Cardiovascular Medicine) 1984,
Martinus Nijhoff Publishers, Boston), and routinely maintained in
McCoy 5A (modified) medium supplemented with 20% foetal bovine
serum, 60 ug/ml penicillin and 100 ug/ml streptomycin and passaged
using trypsin-versene. For experimental work, cells were used
between passage 6 and passage 12 (inclusive). Preequilibrated
NAFION tubes (2.9 mm internal diameter and 25 mm in length) were
incubated with a 40 ug/ml solution of fibronectin (Fn), washed with
PBS and individually placed into sterile-cap polystyrene vials,
then 9 ml of growth medium containing 2.times.10.sup.6 cells was
added to each via. The cell suspension was gassed with a mixture of
5% CO.sub.2 in air and the vial tightly sealed. The vials were then
placed inside a TCP roller bottle and firmly held in a position by
packing. The loaded bottle was then rotated at 1 r.p.m. on a roller
at 37.degree. C. The culture medium was replenished at 24 hr and 72
hr and the tubes removed for subsequent flow testing after 5 days.
Cell growth could be observed through the NAFION tube using a phase
contrast microscope. Having observed that the tubes support cell
attachment and growth over 5 days, the tubes were cultured for 6 hr
in culture medium consisting of Dulbecco's modified Eagle's medium
containing glutamine, 3 mg/l methionine and 25 uCi/ml of
35S-methionine, then further incubated with the normal (McCoy 5A
medium with serum and supplements) medium for a further 15h. The
tubes containing the metabolically-labelled cells were briefly
washed in PBS then inserted into the flow test system as detailed
in FIG. 7. The tubes were subjected to increasing flow rates of a
medium consisting of McCoys 5A medium containing 20 mM Hepes buffer
(pH 7.2) and 20% (v/v) foetal bovine serum at 37.degree. C. for the
specified time periods. Cells released from the tubes were
collected on the downstream glass fibre filters and quantitated by
radioactive determination (liquid scintillation counting).
Following the flow studies, the tube was removed and bisected then
half of the tube was examined for adherent cells by microscopic
techniques and the cells on the other half were removed using
trypsin-versene and the radioactivity in the released cells was
determined
Results
In view of the results with HUAE cells where enhanced cell
spreading, attachment and growth was produced by precoating the
NAFION surface with Fibronectin (Example 3 above), the NAFION tubes
that were used in the flow experiments were precoated with Fn. OCAE
cells seeded into the Fn-coated NAFION tubes attached to the
luminal surface and formed a confluent monolayer of cells during 3
to 5 days of culture. The cells attached to the tube were tested
for cellular attachment in an in vitro flow system that permitted
laminar flow (at flow rates up to 207 ml/min, equivalent to 12.6
dynes/cm.sup.2 shear force, and above this flow rate, turbulent
flow, see FIG. 7 for design). The OCEA cells withstood the shear
force treatments of up to 20 dynes/cm.sup.2, with negligible cell
detachment during the flow treatment (see Table 1 below). The
cellular monolayer was examined microscopically after the flow
treatment and the cells remained attached and well spread to the
luminal surface of the tube, with no evidence of detachment or
damage to the cells. These experiments show that the endothelial
cells form strong attachment to the Fn-coated NAFION tube surface
and can withstand shear forces that are equivalent to those that
would be encountered in vivo.
TABLE 1 ______________________________________ Retention of OCAE
cells on NAFION tubes under fluid flow conditions % cells remaining
% cells detached and Experiment attached to tube recovered on
filters ______________________________________ 1 99.4 0.6 2 99.1
0.9 3 99.0 1.0 4 98.2 1.8 5 97.8 2.2
______________________________________
The cells were cultured on Fibronectin-coated NAFION tubes as
described in the text above, then the cells attached to the tubes
were subjected to the following flow protocol: 10 min at a flowrate
of 66 ml/min corresponding to a shear force of 4 dynes/cm.sup.2
followed by 10 min at 132 ml/min equivalent to 8 dynes/cm.sup.2,
then 10 min at 198 ml/min equivalent to 12 dynes/cm.sup.2, then 10
min at 264 ml/min equivalent to 16 dynes/cm.sup.2, then 10 min at
330 ml/min equivalent to 20 dynes/cm.sup.2. It should be noted that
the increase in flow corresponding to the step going from 12 to 16
dynes/cm.sup.2 necessitated going from laminar to turbulent
flow.
EXAMPLE 5
Studies of in vitro thrombogenicity of NAFION surfaces.
In vitro thrombogenesis of TEFLON, NAFION, tissue culture
polystyrene and vitrogen coated polystyrene surfaces, were studied
in the following manner:
(i) Human Platelet Binding
Human platelets prepared from fresh human plasma were labelled with
Chromium 51. Platelets collected by centrifugation were labelled
with Cr-51 in 0.25 M HEPES/tris buffer pH 7.0 containing a stock
solution of Cr-51 in 0.2 HEPES/tris buffer for 1 hr at room
temperature. The percentage incorporation of Cr-51 into 10
platelets was checked by counting the amount of radioactivity
incorporated in platelets collected by centrifugation and
unincorporated radioactivity in the supernatent. Incorporation of
the label was then inhibited by the addition of 5% ascorbic acid.
Platelets were incubated in the presence of the polymer surface
under study in a 96 well ELISA tray for 3 hours at room
temperature. The polymers were the removed and washed thoroughly in
0.14M NaCl/0.02 HEPES buffer pH 7.0 and bound radioactivity
determined by counting the polymers in a gamma counter. Results
were expressed as numbers of platelets bound to the polymer surface
per mm.sup.2.
(ii) Partial thromboplastin time
Partial thromboplastin time was determined by incubating platelet
poor plasma (Prepared by centrifugation) in the polymer surface in
glass tubes. 0.1M NaCl was added to the plasma and the time for
clot formation measured. This time was taken as the partial
thromboplastin time.
Results
The platelet binding experiments showed that polystyrene, TEFLON
and NAFION bound between approximately 12,000 and 18,000 platelets
per mm.sup.2 whereas polystyrene coated with vitrogen 100 (purified
collagen) bound 163,682 platelets per mm.sup.2. Actual figures
obtained were for polystyrene 15,023; for TEFLON 17,860 and for
NAFION 18,070 platelets per mm.sup.2. Partial thromboplastin times
revealed that NAFION required 210 seconds for clot formation
followed by vitrogen 100-coated polystyrene that required 238s;
TEFLON 247 s and polystyrene 250 s.
These data indicate that NAFION and TEFLON have similar partial
thromboplastin times and platelet binding properties (low, compared
to collagen surfaces) which suggests that NAFION is no more
thrombogenic than TEFLON.
EXAMPLE 6
Porous NAFION Implants
The use of autograft material is still the most desirable method
used to replace diseased or damaged tissues or organs. However, due
to anatomical or other considerations, such as those of infection
or rejection, this approach may not be feasible. Of particular
challenge is the repair of connective tissue defects. Both
naturally derived and synthetic materials have been used in this
regard, for example, injectable solubilized collagen and polymeric
hydrogels. There is increasing interest in the provision of
synthetic materials as components of prosthetic devices.
Methods
NAFION implants were prepared by mixing a 5% solution of NAFION
Equivalent Weight 1100 with NaCl crystals in approximately 10:1
ratio. The mixture was poured into either glass petri dishes or
small (20 ml) volume beakers and incubated at 60.degree. C. for
between four and seven days. After this time the NaCl was dissolved
in distilled water. However, implants could have been made by
alternative techniques described in the art, e.g. sintering,
thermal expansion, laser or ion beam drilling, etc. The material
samples were sterilized prior to implantation using an industrial
method of ethylene oxide processing. In vivo biocompatibility
testing was conducted using males of an inbred strain of BALB/c
mice. Animals were anaesthetised using ether and their dorsal and
flank regions prepared by clipping the fur and swabbing with
HIBITANE disinfectant (10% in 70% ethanol, each (v/v)). Implants
were inserted subdermally by making a small incision with a pair of
scissors and further blunt dissection to prepare a small pocket
into which each implant was placed. The skin was closed using two
sutures (Mersilk 4.0) and swabbed again with HIBITANE. Each animal
received one implant of the material and was caged separately after
surgery. Animals were biopsied after one, three, four, six and
twelve weeks. At biopsy, implants were examined macroscopically for
signs of lysis or gross inflammation, excised with the overlying
skin and fixed in 10% formaldehyde in normal saline. The tissue was
dehydrated in ethanol and prepared for routine histology by
embedding in Historesin (LKB); 2 mm sections were stained with
haematoxylin and eosin and viewed in an Olympus BH-2
microscope.
Results
All implants were recovered progressively up to 12 weeks.
Macroscopic examination revealed that no overt tissue inflammation
was present. An indication of the resistance of the implant to
cellular degradation was that the original angular contours of the
cut edges of the implants were still visible even after 12 weeks in
vivo. Implants appeared to maintain their original size throughout
the 12-week study period. Histological examination showed that
after one week, implants were infiltrated with lymphocytes and a
highly cellular connective tissue capsule had formed around the
periphery. Lymphocytes had also migrated into the centre of the
implants. Three and four week implants appeared similar. Peripheral
aspects of the implants were well embedded in the fibrous tissue
surrounding the implant. Some fibrous tissue ingrowth was seen
particularly after four weeks, as was the presence of large
capillaries. At this stage multinuclear giant cells were seen in
close association with the implants. After six weeks cellular and
fibrous tissue ingrowth was well developed. The lymphocyte
inflammatory response was reduced by this time however more
multinuclear giant cells were found around the edges of the
implants. Twelve weeks after implantation, isolated fingers of
fibrous tissue had extended into the centre of most of the
implants. These were populated by fibroblasts and lymphocytes and
contained small blood capillaries. All implants were well embedded
in host tissue and larger blood vessels were observed growing
across the surface of the implants. The implants showed no sign of
material degradation.
In summary Examples 1 to 5 demonstrate the excellent cell response
to NAFION in different forms. By successfully growing a number of
mammalian cell types, notably human arterial endothelial cells, on
a variety of NAFION substrates we have shown that NAFION has good
cell supportive characteristics. In particular, we have
demonstrated the efficacy of NAFION coated TEFLON for support of
endothelial cell growth, and that endothelial cells grown on NAFION
tubes by the procedures contained in this invention resist shear
forces due to fluid flow. It is generally understood that a
covering of only approximately 20% of the luminal surface of a
vascular graft by metabolising endothelial cells is required to
avoid thrombogenesis and we have shown that a morphologically
normal endothelial surface is achieved quickly on NAFION. This is
in contrast to attempts to culture endothelial cells on TEFLON
where cells remain fibroblastoid until reaching confluence and are
poorly attached to the surface. Example 6 demonstrates the
biological acceptance of porous NAFION implants indicating that
NAFION may be useful for connective tissue or soft tissue
prostheses.
These results suggest that NAFION or indeed any copolymer of
perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a
monomer, may be used for the in vitro attachment and growth of
animal cells, and may be incorporated into a vascular prosthesis or
be a useful alternative to commercially available materials
currently used as components of vascular prostheses. Preferably,
the copolymer of the present invention may be readily cast into
tubes or coated onto pre-existing tubes to serve as an effective
vascular graft. The effectiveness of the said copolymer as a
component of a vascular graft may be further enhanced by the many
apparent variations in its preparation as discussed with reference
to NAFION herein. The use of the said copolymer as a surface for
endothelial cell attachment and growth may be of particular value
in the new technique of cell seeding of vascular grafts and
prostheses prior to implantation.
The foregoing describes only some embodiments of the present
invention and modifications obvious to those skilled in the art,
can be made thereto without departing from the scope and ambit of
the invention.
* * * * *